Antitumor drugs and radiation therapy work, to a significant degree, by activation of tumor cell apoptosis (Reed, J C. et al. J. Cell. Biochem 60:23-32, 1996). Therapeutic efficacy is therefore dependent on inherent mechanisms that regulate susceptibility to apoptosis. Apoptosis is regulated by at least two pathways; mitochondrial pathways and membrane death receptors (Green D R., Science 281:1309-12, 1998, Ashkenazi A., Curr Opin Cell Bio 11:225-260, 1999, Gross, A. et al . Genes Dev 13:1899-911, 1999). Mitochondrial release of cytochrome c into the cytosol activates procaspase-9 with resulting activation of downstream effector caspases and cellular apoptosis. Activated caspases are responsible for the proteolytic events important in mediation of apoptosis. Several antiapoptotic proteins, including Bcl-2, Bcl-XL, or Bcl-W, inhibit mitochondrial release of cytochrome c and prevent alteration of the megachannel (Green D R., Science 281:1309-12, 1998, Li, P., Cell 91:479-89, 1997, Yang, J., Science 275:1129-32, 1997, Tsujimoto, Y., FEBS Lett 466:6-10, 2000).
Thus anti-apoptotic proteins can stabilize mitochondrial membrane integrity, prevent cytochrome release, and block apoptosis. These molecular events contribute to the intrinsic tumor resistance to cytotoxic drugs and radiation (Sun Y., Cancer Res 62:6323-28, 2002, Violette, S., Int. J. Cancer 98:498-504, 2002, Amundson, SA., Cancer Res 60:6101-10, 2000). In vivo studies have confirmed these concepts, showing that high Bcl-2, and/or Bcl-XL are predictive of a poor prognosis in patients with colon cancer (Ogura, E., Oncol Rep 6:365-9, 1999, and Maurer Calif., Dig Dis Sci 43:2641-8, 1998). Bcl-2-mediated inhibition of in vivo apoptosis may also prevent immunogenicity of tumors, thus contributing to a progressive, metastatic and lethal phenotype of otherwise regressive clonal variants that arise during tumor growth (Bonnotte, B., J of Immuno. 161:1433-38, 1998). Thus one component of the persistence/resistance of tumor cells with increased Bcl-2 expression may reflect a reduced capacity to induce a host immune response.
Bcl-2 can be upregulated after treatment with prostaglandins (PGE2) in tumor cells (Sheng, H., Cancer Res 58:362-366, 1998). COX-2 mediated production of PGE2 may contribute to tumor resistance, in part, by regulating anti-apoptotic gene expression. Yet, Bcl-2 levels are maintained in cells treated with NSAIDs, suggesting that up-regulation of Bcl-2 can occur independently of PGE2 (Sun Y., Cancer Res 62:6323-28, 2002). Increased expression of Bcl-2, and Bcl-XL are noted in colorectal tumors compared with histologically normal mucosa (Sinicrope F A., Cancer Res 55:237-41, 1995). Thus factors that are responsible for the up-regulation of Bcl-2 family genes in tumors appear to represent a promising and yet unexplored therapeutic avenue.
One kinase responsible for protection against apoptosis in tumor cells may be Akt/protein kinase B. Akt, also referred to as Rac regulate the balance between survival and apoptosis by several mechanisms. Akt is activated by a variety of growth factors (Burgering B T., et al. Nature 376:599-602, 1995, Franke T F., et al. Cell 81:727-36, 1995) and acts in a LY294002 (inhibitor of PI3K) and Wortmannin-sensitive pathway. Akt is activated by phospholipid binding and phosphorylation localized to at least two sites (Thr308 and Ser473). Activated Akt inactivates by phosphorylation several targets, including caspase 9, Bad, GSK3, and members of the forkhead transcription factor family. Akt overexpression has been shown to be a critical and early event during sporadic colon carcinogenesis (Roy, H K., et al Carcinogenesis 23:201-5, 2002). Furthermore, phosphorylation of Akt is required for suppression of apoptosis and tumor progression of human colorectal carcinoma (Itoh N., et al Cancer 94:3127-34, 2002). Itoh showed that expression of phosphorylated Akt (Ser473) by immunohistochemistry was correlated with depth of invasion, lymph node metastasis, and advanced tumor stage in human colorectal. Correlation with Bcl-2 expression nearly reached statistical significance as well. In this study, increased phosphorylated Akt expression was also directly correlated with an increasing growth index and a decreased apoptotic index in human tumor tissue resection specimens. Direct inhibition of the Akt pathway, such as with LY294002, may have considerable toxicity given the potential physiologic role in normal cell senescence and differentiation in the colorectal mucosa (Alessi D R., et al. Curr Biol 7:261-69, 1997, Klippel A., et al Mol Cell Biol 17:338-44, 1997) as well as other tissues. This makes determination and selective targeting of relevant tumor upstream activators of the Akt pathway theoretically preferable.
The human Reg gene family of ligands consists of four secreted and structurally unique protein that share sequence similarity with the carbohydrate-binding domain of C-type lectins (FIG. 1). The initial cDNA in this gene family was named Reg for its role in islet of Langerhans regeneration following partial pancreatectomy (now known as Reg Iα). Additional members of the human Reg gene family are regenerating gene homologue (Reg Iβ) and pancreatitis-associated protein (Reg III). All are constitutively expressed in the normal proximal gastrointestinal tract. While the function of this gene family is poorly understood, recent data has suggested that Reg family members may function as tissue mitogens. Reg Iα is mitogenic for gastric mucosal cells (Fukui H., et al. Gastro 115:1483-93, 1998), and pancreatic ductal and beta cells (Zenilman, Me., et al. Gastroenterology 110:1208-14, 1996, Zenilman, Me., et al. Pancreas 17:256-61, 1998, Watanabe, T., PNAS 91:3589-92, 1994). The serum concentration of Reg Iα is significantly increased in many gastrointestinal malignancies, including gastric and pancreatic adenocarcinoma (Satomura, Y., et al. J Gastroenterology 30:643-50, 1995). For patients with early stage colonic adenocarcinoma undergoing surgical resection, Reg Iα mRNA expression alone or co-expression of Reg Iα and Reg III mRNA by the carcinoma had an adverse affect on disease free survival that was independent of tumor stage or site (Macadam, RCA., et al British J Cancer 83:188-95, 2000).
We recently identified a novel member of this gene family, Reg IV, which has significant constitutive expression in the distal gastrointestinal tract (Hartupee J C., et al BBA 1518:287-93, 2001). We showed by molecular modeling, that the Reg IV protein showed maintenance of the conserved contact surface residues that cluster on a single face of the 3-dimensional molecule present in all other members of the Reg gene family (FIGS. 1 and 2). This suggests that Reg proteins may share similar physiologic actions. Reg IV is of considerable interest because of its possible role, along with other members of the Reg gene family in the pathogenesis of colorectal adenocarcinoma. Reg IV is overexpressed by a majority of colorectal adenocarcimonas. By differential display, Reg IV was among several genes with increased mRNA expression in several colon cancer cell lines selected for increased in vitro resistance to a cancer chemotherapeutic agent, 5-FU (Violette S et al, Int J. Cancer 103:185-193).
Expression of many genes is increased or decreased in gastrointestinal malignancy as compared with the normal gastrointestinal mucosa. This correlation does not provide a causative association (e.g., apoptosis resistance or malignant phenotype). In fact most differentially regulated genes will not serve an integral role in the malignancy process or offer a currently recognizable therapeutic intervention. There is a need in the art to identify genes that do serve an integral role in the malignancy process and which will offer therapeutic interventions.